The long term goal of the proposed screening project is the identification of novel inhibitors of the integrin IIb 3 with more favorable characteristics than currently available agents. The first specific aim is the identification of additional novel antagonists via high throughput screening, the second is the assessment of their specificity for IIb 3, and the third is the characterization of their mechanisms of inhibition. The development of new inhibitors represents a significant clinical need because current IIb 3 antagonists have been shown to cause thrombocytopenia in some patients, and the orally active IIb 3 inhibitors paradoxically caused increased mortality in Phase III trials. All of the orally active agents, as well as the 2 FDA-approved small molecule parenteral agents, function by competitively blocking the ligand binding site of IIb 3; thus, these agents act as ligand-mimetics. It is known that the binding of ligand by IIb 3 results in conformational changes in the receptor extracellular structure, and that these changes expose neo-epitopes on the receptor known as ligand-induced binding sites (LIBS). These conformational changes may underlie both the thrombocytopenia and the paradoxical prothrombotic effects of the oral agents. Thus, IIb 3 antagonists that lack ligand-mimetic activity may improve both therapeutic safety and efficacy. Specific Aim 1: To identify novel inhibitors of platelet adhesion to fibrinogen using platelet adhesion to fibrinogen as the screening assay. We have already used this assay to screen more than 33,000 compounds. Specific Aim 2: To assess the specificity of any inhibitors identified in the primary screen by testing their effects on various other cell adhesion molecules including but not limited to: interaction of the highly homologous integrin V 3 with vitronectin, interaction of integrin 2 1 with collagen, and interaction of GPIb with von Willebrand factor. The effect of the compounds on cell viability will also be assessed. Specific Aim 3: To characterize the mechanism by which the compounds inhibit adhesion of platelets to fibrinogen. The following assays will be employed to determine whether compounds interact directly with IIb 3: 1) aggregation of platelets in plasma following the addition of exogenous activators; 2) binding of fibrinogen to purified IIb 3; 3) adhesion of cells expressing IIb 3 to immobilized fibrinogen; 4) binding of soluble fibrinogen to platelets following treatment with IIb 3 activators. Those compounds that interact directly with IIb 3 will be characterized further to assess their effects on the conformation of IIb 3 by 1) measuring the exposure of ligand-induced binding site (LIBS) epitopes on platelets, and 2) testing the ability of transient exposure of the compounds to "prime" purified IIb 3 so that it binds fibrinogen. Finally, compounds that inhibit IIb 3 but do not induce conformational changes in the receptor will be selected for additional in silico docking studies, in vivo testing in animal models of thrombosis and hemostasis, and chemical modifications as a prelude to potential therapeutic development. [unreadable] [unreadable] [unreadable]